Revised Structure of Anthelvencin A and Characterization of the Anthelvencin Biosynthetic Gene Cluster.

Anthelvencins A and B are pyrrolamide metabolites produced by Streptomyces venezuelae ATCC 14583 and 14585. Isolated in 1965, they were reported to exhibit anthelmintic and moderate antibacterial activities. In this study, we revise the structure of anthelvencin A and identify a third anthelvencin metabolite, bearing two N-methylated pyrrole groups, which we named anthelvencin C. We sequenced the genome of S. venezuelae ATCC 14583 and identified a gene cluster predicted to direct the biosynthesis of anthelvencins. Functional analysis of this gene cluster confirmed its involvement in anthelvencin biosynthesis and allowed us to propose a biosynthetic pathway for anthelvencins. In addition to a nonribosomal peptide synthetase (NRPS), the assembly of anthelvencins involves an enzyme from the ATP-grasp ligase family, Ant23. We propose that Ant23 uses a PCP-loaded 4-aminopyrrole-2-carboxylate as substrate. As observed for the biosynthesis of the other pyrrolamides congocidine (produced by Streptomyces ambofaciens ATCC 25877) and distamycin (produced by Streptomyces netropsis DSM 40846), the NRPS assembling anthelvencins is composed of stand-alone domains only. Such NRPSs, sometimes called type II NRPSs, are less studied than the classical multimodular NRPSs. Yet, they constitute an interesting model to study protein-protein interactions in NRPSs and are good candidates for combinatorial biosynthesis approaches.

Complete Genome Sequence of Streptomyces sp. TN58, a Producer of Acyl Alpha-l-Rhamnopyranosides.

Streptomyces sp. TN58, isolated from a Tunisian soil sample, produces several natural products, including acyl alpha-l-rhamnopyranosides. It possesses a 7.6-Mb linear chromosome. This is, to our knowledge, the first genome sequence of a microorganism known to produce acyl alpha-l-rhamnopyranosides, and it will be helpful to study the biosynthesis of these specialized metabolites.

Draft Genome Sequence of Streptomyces sp. M1013, a Close Relative of Streptomyces ambofaciens and Streptomyces coelicolor.

We report the draft genome sequence of Streptomyces sp. M1013, a strain isolated from the Medicago arborea rhizosphere in Izmir, Turkey. An average nucleotide identity (ANI) analysis reveals that this strain belongs to the same species as Streptomyces canus ATCC12647 and is closely related to Streptomyces ambofaciens and Streptomyces coelicolor.

Complete genome sequence of Streptomyces ambofaciens ATCC 23877, the spiramycin producer.

Streptomyces ambofaciens ATCC23877 is a soil bacterium industrially exploited for the production of the macrolide spiramycin which is used in human medicine as an antibacterial and anti-toxoplasmosis chemical. Its genome consists of a 8.3 Mbp linear chromosome and a 89 kb circular plasmid. The complete genome sequence reported here will enable us to investigate Streptomyces genome evolution and to discover new secondary metabolites with potential applications notably in human medicine.

Natural combinatorial biosynthesis involving two clusters for the synthesis of three pyrrolamides in Streptomyces netropsis.

The pyrrolamides constitute a small family of secondary metabolites that are known for their ability to bind noncovalently to the DNA minor groove with some sequence specificity. To date, only a single pyrrolamide biosynthetic gene cluster has been reported, directing the synthesis of congocidine (netropsin) in Streptomyces ambofaciens. In this study, we improve our understanding of pyrrolamide biosynthesis through the identification and characterization of the gene cluster responsible for the production of distamycin in Streptomyces netropsis DSM40846. We discover that the strain produces two other pyrrolamides, the well-characterized congocidine and a congocidine/distamycin hybrid that we named disgocidine. S. netropsis DSM40846 genome analysis led to the identification of two distinct pyrrolamide-like biosynthetic gene clusters. We show here that these two clusters are reciprocally dependent for the production of the three pyrrolamide molecules. Furthermore, based on detailed functional analysis of these clusters, we propose a biosynthetic route to congocidine and distamycin and an updated model for pyrrolamide assembly. The synthesis of disgocidine, the distamycin/congocidine hybrid, appears to constitute the first example of "natural combinatorial biosynthesis" between two related biosynthetic pathways. Finally, we analyze the genomic context of the two biosynthetic gene clusters and suggest that the presently interdependent clusters result from the coevolution of two ancestral independent pyrrolamide gene clusters.

Organization of the biosynthetic gene cluster for the macrolide antibiotic spiramycin in Streptomyces ambofaciens.

Spiramycin, a 16-membered macrolide antibiotic used in human medicine, is produced by Streptomyces ambofaciens; it comprises a polyketide lactone, platenolide, to which three deoxyhexose sugars are attached. In order to characterize the gene cluster governing the biosynthesis of spiramycin, several overlapping cosmids were isolated from an S. ambofaciens gene library, by hybridization with various probes (spiramycin resistance or biosynthetic genes, tylosin biosynthetic genes), and the sequences of their inserts were determined. Sequence analysis showed that the spiramycin biosynthetic gene cluster spanned a region of over 85 kb of contiguous DNA. In addition to the five previously described genes that encode the type I polyketide synthase involved in platenolide biosynthesis, 45 other genes have been identified. It was possible to propose a function for most of the inferred proteins in spiramycin biosynthesis, in its regulation, in resistance to the produced antibiotic or in the provision of extender units for the polyketide synthase. Two of these genes, predicted to be involved in deoxysugar biosynthesis, were inactivated by gene replacement, and the resulting mutants were unable to produce spiramycin, thus confirming their involvement in spiramycin biosynthesis. This work reveals the main features of spiramycin biosynthesis and constitutes a first step towards a detailed molecular analysis of the production of this medically important antibiotic.

Intraspecific variability of the terminal inverted repeats of the linear chromosome of Streptomyces ambofaciens.

The sequences of the terminal inverted repeats (TIRs) ending the linear chromosomal DNA of two Streptomyces ambofaciens strains, ATCC23877 and DSM40697 (198 kb and 213 kb, respectively), were determined from two sets of recombinant cosmids. Among the 215 coding DNA sequences (CDSs) predicted in the TIRs of strain DSM40697, 65 are absent in the TIRs of strain ATCC23877. Reciprocally, 45 of the 194 predicted CDSs are specific to the ATCC23877 strain. The strain-specific CDSs are located mainly at the terminal end of the TIRs. Indeed, although TIRs appear almost identical over 150 kb (99% nucleotide identity), large regions of DNA of 60 kb (DSM40697) and 48 kb (ATCC23877), mostly spanning the ends of the chromosome, are strain specific. These regions are rich in plasmid-associated genes, including genes encoding putative conjugal transfer functions. The strain-specific regions also share a G+C content (68%) lower than that of the rest of the genome (from 71% to 73%), a percentage that is more typical of Streptomyces plasmids and mobile elements. These data suggest that exchanges of replicon extremities have occurred, thereby contributing to the terminal variability observed at the intraspecific level. In addition, the terminal regions include many mobile genetic element-related genes, pseudogenes, and genes related to adaptation. The results give insight into the mechanisms of evolution of the TIRs: integration of new information and/or loss of DNA fragments and subsequent homogenization of the two chromosomal extremities.

Evolution of the terminal regions of the Streptomyces linear chromosome.

Comparative analysis of the Streptomyces chromosome sequences, between Streptomyces coelicolor, Streptomyces avermitilis, and Streptomyces ambofaciens ATCC23877 (whose partial sequence is released in this study), revealed a highly compartmentalized genetic organization of their genome. Indeed, despite the presence of specific genomic islands, the central part of the chromosome appears highly syntenic. In contrast, the chromosome of each species exhibits large species-specific terminal regions (from 753 to 1,393 kb), even when considering closely related species (S. ambofaciens and S. coelicolor). Interestingly, the size of the central conserved region between species decreases as the phylogenetic distance between them increases, whereas the specific terminal fraction reciprocally increases in size. Between highly syntenic central regions and species-specific chromosomal parts, there is a notable degeneration of synteny due to frequent insertions/deletions. This reveals a massive and constant genomic flux (from lateral gene transfer and DNA rearrangements) affecting the terminal contingency regions. We speculate that a gradient of recombination rate (i.e., insertion/deletion events) toward the extremities is the force driving the exclusion of essential genes from the terminal regions (i.e., chromosome compartmentalization) and generating a fast gene turnover for strong adaptation capabilities.

Disruption of sblA in Streptomyces lividans permits expression of a heterologous alpha-amylase gene in the presence of glucose.

In a transposition mutant of Streptomyces lividans TK24, the usually glucose-repressible expression of a heterologous alpha-amylase gene (aml) became resistant to glucose repression. The transposon had inserted into an ORF called sblA which encodes a 274 aa product sharing significant sequence similarities with various phosphatases that act on small phosphorylated substrates. sblA was transcribed as a monocistronic mRNA and its transcription was enhanced at the transition phase. Because its transcriptional and putative translational start points coincide, sblA is likely to be translated in the absence of a conventional RBS. The sblA-disrupted mutant is characterized by early growth arrest in glucose-grown cultures and by partial relief of glucose repression of aml expression.